O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification (PTM) that involves the addition of a single sugar, N-acetylglucosamine, to serine or threonine residues of protein in the cytoplasm, nucleus, or mitochondria. Two enzymes facilitate this modification: O-GlcNAc transferase (OGT), which adds the modification and O-GlcNAcase (OGA), which removes the modification. The addition and removal of O-GlcNAc by these two enzymes is termed O-GlcNAc cycling and works as a molecular switch. Of note, PTMs are used in regulating protein function and therefore can affect mitochondrial activity. We postulate that O-GlcNAc regulates mitochondrial function. The proper regulation of mitochondrial function is essential for cellular metabolism and respiration. Improper regulation of mitochondria leads to cellular damage and disease development. Here, we examined how elevation of O-GlcNAc affects mitochondrial function. In order to elevate O-GlcNAc levels, we supplemented SH-SY5Y and NT2 neuroblastoma cells with Thiamet-G (TMG), an OGA inhibitor, or Glucosamine (GlcN), which increases OGT substrate availability. We found that both TMG and GlcN treatment increased O-GlcNAc levels and OGA protein and transcript levels. Next, we found that mitochondrial respiration was altered and ATP production was decreased. We found that reactive oxygen species (ROS) production was significantly reduced in treated cells. We also found that pathway activity and protein expression of the master regulator of antioxidant response, the transcription factor NRF2 was down regulated. Finally, the protein expression of NRF2 antioxidant genes such as manganese superoxide dismustase (MnSOD) and thioredoxin reductase 1 (TXNRD1) was lower. Altogether, these data demonstrate that prolonged alterations to the cellular homeostasis of O-GlcNAc affects mitochondrial function by reprogramming metabolic activity. Further understanding of how O-GlcNAc cycling regulates metabolism will provide new insights into metabolic diseases such as Alzheimers.